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This figure shows the proliferation and phneotype of tumor lines in response to NECA.

Supplementary Figure 2

This supplementary Figure shows the phenotype and function of CD8+ T cells and tumor cells following A2A blockade single agent therapy.

This supplementray Figure shows the expression of IFNy and Granzyme B on tumor-infiltrating lymphocytes 7 days post anti-PD-1/ anti-PD-1/A2A blockade.

Supplementary Figure 5

This Supplementary Figure shows the expression of CD73 and adenosine receptors by tumor lines.

This document contains the Figure Legends for the Supplementary data Figures.

This Supplememntary Figure shows the effect of combined PD-1 and A2A blockade on in vitro effector functions of OT-I cells.

This supplementary Figure shows the expression of A2A and IFNy on tumor infiltrating lymphocytes following PD-1 blockade.

This supplementary figure shows the phenotype of tumor-infiltrating lymphocytes following PD-1 or PD-1/A2A blockade.

Supplementary Figure 1

Immunotherapy is widely accepted as a powerful new treatment modality for the treatment of cancer. The most successful form of immunotherapy to date has been the blockade of the immune checkpoints PD-1 and CTLA-4. Combining inhibitors of both PD-1 and CTLA-4 increases the proportion of patients who respond to immunotherapy. However, most patients still do not respond to checkpoint inhibitors, and prognostic biomarkers are currently lacking. Therefore, a better understanding of the mechanism by which these checkpoint inhibitors enhance antitumor immune responses is required to more accurately predict which patients are likely to respond and further enhance this treatment modality. Our current study of two mouse tumor models revealed that CD4+Foxp3− cells activated by dual PD-1/CTLA-4 blockade modulated the myeloid compartment, including activation of conventional CD103+ dendritic cells (DC) and expansion of a myeloid subset that produces TNFα and iNOS (TIP-DCs). CD4+Foxp3− T cell–mediated activation of CD103+ DCs resulted in enhanced IL12 production by these cells and IL12 enhanced the therapeutic effect of dual PD-1/CTLA-4 blockade. Given the importance of these myeloid subsets in the antitumor immune response, our data point to a previously underappreciated role of CD4+Foxp3− cells in modulating this arm of the antitumor immune response. Cancer Immunol Res; 6(9); 1069–81. ©2018 AACR.

Supplementary Figure 6

Supplementary Figure 3

Immunotherapy is rapidly emerging as a cancer treatment with high potential. Recent clinical trials with anti-CTLA-4 and anti–PD-1/PD-L1 antibodies (mAbs) suggest that targeting multiple immunosuppressive pathways may significantly improve patient survival. The generation of adenosine by CD73 also suppresses antitumor immune responses through the activation of A2A receptors on T cells and natural killer (NK) cells. We sought to determine whether blockade of A2A receptors could enhance the efficacy of anti–PD-1 mAb. The expression of CD73 by tumor cells limited the efficacy of anti–PD-1 mAb in two tumor models, and this was alleviated with concomitant treatment with an A2A adenosine receptor antagonist. The blockade of PD-1 enhanced A2A receptor expression on tumor-infiltrating CD8+ T cells, making them more susceptible to A2A-mediated suppression. Thus, dual blockade of PD-1 and A2A significantly enhanced the expression of IFNγ and Granzyme B by tumor-infiltrating CD8+ T cells and, accordingly, increased growth inhibition of CD73+ tumors and survival of mice. The results of our study indicate that CD73 expression may constitute a potential biomarker for the efficacy of anti–PD-1 mAb in patients with cancer and that the efficacy of anti–PD-1 mAb can be significantly enhanced by A2A antagonists. We have therefore revealed a potentially novel biomarker for the efficacy of anti–PD-1 that warrants further investigation in patients. Because our studies used SYN-115, a drug that has already undergone phase IIb testing in Parkinson disease, our findings have immediate translational relevance for patients with cancer. Cancer Immunol Res; 3(5); 506–17. ©2015 AACR.

This supplementary figure shows the expression of PD-1 on tumor infiltrating immune subsets.

Supplementary Figure 1 - PDF file 73K, Tumor cells express high levels of PD-L1

Supplementary Figure 2 - PDF file 20K, Expression of PD-1 is upregulated on CAR T cells following antigen stimulation with e0771-Her2+ tumor cells

upplementary Figure 3 - PDF file 17K, Gr1+ cells express PD-1 and PD-L1

Purpose: To determine the antitumor efficacy and toxicity of a novel combination approach involving adoptive T-cell immunotherapy using chimeric antigen receptor (CAR) T cells with an immunomodulatory reagent for blocking immunosuppression.Experimental Design: We examined whether administration of a PD-1 blocking antibody could increase the therapeutic activity of CAR T cells against two different Her-2+ tumors. The use of a self-antigen mouse model enabled investigation into the efficacy, mechanism, and toxicity of this combination approach.Results: In this study, we first showed a significant increase in the level of PD-1 expressed on transduced anti-Her-2 CD8+ T cells following antigen-specific stimulation with PD-L1+ tumor cells and that markers of activation and proliferation were increased in anti-Her-2 T cells in the presence of anti-PD-1 antibody. In adoptive transfer studies in Her-2 transgenic recipient mice, we showed a significant improvement in growth inhibition of two different Her-2+ tumors treated with anti-Her-2 T cells in combination with anti-PD-1 antibody. The therapeutic effects observed correlated with increased function of anti-Her-2 T cells following PD-1 blockade. Strikingly, a significant decrease in the percentage of Gr1+ CD11b+ myeloid-derived suppressor cells (MDSC) was observed in the tumor microenvironment of mice treated with the combination therapy. Importantly, increased antitumor effects were not associated with any autoimmune pathology in normal tissue expressing Her-2 antigen.Conclusion: This study shows that specifically blocking PD-1 immunosuppression can potently enhance CAR T-cell therapy that has significant implications for potentially improving therapeutic outcomes of this approach in patients with cancer. Clin Cancer Res; 19(20); 5636–46. ©2013 AACR.

Supplementary Figure Legends - PDF file 66K, Supplementary Figure legends

Supplementary Figure S1. Expression of activation and memory markers on transduced mouse T cells. Supplementary Figure S2. CAR T cell cytotoxic responses are not modulated by anti-4-1BB mAb therapy. Supplementary Figure S3. Her2 expression level on the mouse breast cancer cell line e0771-Her2 is comparable to the Her2-overexpressing human breast cancer cell line SKBR3. Supplementary Figure S4. Tumor weight is significantly reduced following CAR T cell and alpha-4-1BB therapy. Supplementary Figure S5. Anti-4-1BB therapy does not modulate the frequency of transferred or endogenous CD8+ tumor-infiltrating T cells. Supplementary Figure S6. Anti-4-1BB therapy modulates the frequency of mature dendritic cells in tumors and draining lymph nodes. Supplementary Figure S7. Anti-4-1BB does not modulate the frequency of MDSCs and DCs in RAG -/- mice. Supplementary Figure S8. Anti-4-1BB therapy drives maturation of NK cells.

Adoptive immunotherapy utilizing chimeric antigen receptor (CAR) T cells has demonstrated high success rates in hematologic cancers, but results against solid malignancies have been limited to date, due in part to the immunosuppressive tumor microenvironment. Activation of the 4-1BB (CD137) pathway using an agonistic α-4-1BB antibody is known to provide strong costimulatory signals for augmenting and diversifying T-cell responses. We therefore hypothesized that a combination of α-4-1BB and CAR T-cell therapy would result in improved antitumor responses. Using a human-Her2 self-antigen mouse model, we report here that α-4-1BB significantly enhanced CAR T-cell efficacy directed against the Her2 antigen in two different established solid tumor settings. Treatment also increased the expression of IFNγ and the proliferation marker Ki67 in tumor-infiltrating CAR T cells when combined with α-4-1BB. Strikingly, α-4-1BB significantly reduced host immunosuppressive cells at the tumor site, including regulatory T cells and myeloid-derived suppressor cells, correlating with an increased therapeutic response. We conclude that α-4-1BB has a multifunctional role for enhancing CAR T-cell responses and that this combination therapy has high translational potential, given current phase I/II clinical trials with α-4-1BB against various types of cancer. Cancer Res; 77(6); 1296–309. ©2017 AACR.

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Oncolytic virotherapy using vaccinia virus (Vv) has shown some encouraging antitumor responses in mouse models and patients, but the breadth of efficacy in clinical trials has been somewhat limited. Given that antitumor effects have correlated with increased host immune responses, we hypothesized that improved therapeutic outcomes may be achieved by using oncolytic virus (OV) in combination with a potent immune agonist reagent. In this study, we carried out a preclinical evaluation of a genetically engineered strain of oncolytic vaccinia virus (Vvdd) for its capacity to induce antitumor responses when combined with an agonist antibody (Ab) specific for the costimulatory molecule 4-1BB (CD137). In immune-competent syngeneic mouse models of cancer, this combination therapy significantly reduced the growth of established subcutaneous tumors relative to either treatment alone. Importantly, the development of pulmonary metastatic lesions was also reduced. Tumor growth inhibition was associated with increased numbers of CD11b+ and CD11c+ myeloid cells in the tumor draining lymph nodes, greater infiltration of CD8+ effector T and natural killer (NK) cells, and a more sustained presence of neutrophils at the tumor site. Depletion of T or NK cells or neutrophils reduced efficacy, confirming their contribution to an effective therapeutic response. We further extended this conclusion through results from IFNγ-deficient mice. In summary, our findings offered a proof-of-concept for a combinatorial approach to enhance the antitumor efficacy of an OV, suggesting a strategy to improve their use as an immunotherapeutic treatment for cancer. Cancer Res; 72(7); 1651–60. ©2012 AACR.

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Immunotherapy is widely accepted as a powerful new treatment modality for the treatment of cancer. The most successful form of immunotherapy to date has been the blockade of the immune checkpoints PD-1 and CTLA-4. Combining inhibitors of both PD-1 and CTLA-4 increases the proportion of patients who respond to immunotherapy. However, most patients still do not respond to checkpoint inhibitors, and prognostic biomarkers are currently lacking. Therefore, a better understanding of the mechanism by which these checkpoint inhibitors enhance antitumor immune responses is required to more accurately predict which patients are likely to respond and further enhance this treatment modality. Our current study of two mouse tumor models revealed that CD4+Foxp3− cells activated by dual PD-1/CTLA-4 blockade modulated the myeloid compartment, including activation of conventional CD103+ dendritic cells (DC) and expansion of a myeloid subset that produces TNFα and iNOS (TIP-DCs). CD4+Foxp3− T cell–mediated activation of CD103+ DCs resulted in enhanced IL12 production by these cells and IL12 enhanced the therapeutic effect of dual PD-1/CTLA-4 blockade. Given the importance of these myeloid subsets in the antitumor immune response, our data point to a previously underappreciated role of CD4+Foxp3− cells in modulating this arm of the antitumor immune response. Cancer Immunol Res; 6(9); 1069–81. ©2018 AACR.

Adoptive immunotherapy utilizing chimeric antigen receptor (CAR) T cells has demonstrated high success rates in hematologic cancers, but results against solid malignancies have been limited to date, due in part to the immunosuppressive tumor microenvironment. Activation of the 4-1BB (CD137) pathway using an agonistic α-4-1BB antibody is known to provide strong costimulatory signals for augmenting and diversifying T-cell responses. We therefore hypothesized that a combination of α-4-1BB and CAR T-cell therapy would result in improved antitumor responses. Using a human-Her2 self-antigen mouse model, we report here that α-4-1BB significantly enhanced CAR T-cell efficacy directed against the Her2 antigen in two different established solid tumor settings. Treatment also increased the expression of IFNγ and the proliferation marker Ki67 in tumor-infiltrating CAR T cells when combined with α-4-1BB. Strikingly, α-4-1BB significantly reduced host immunosuppressive cells at the tumor site, including regulatory T cells and myeloid-derived suppressor cells, correlating with an increased therapeutic response. We conclude that α-4-1BB has a multifunctional role for enhancing CAR T-cell responses and that this combination therapy has high translational potential, given current phase I/II clinical trials with α-4-1BB against various types of cancer. Cancer Res; 77(6); 1296–309. ©2017 AACR.

International audience; While adoptive immunotherapy using chimeric antigen receptor (CAR)-modified T cells can induce remission of some tumors, the role of other CAR-modified leukocytes is not well characterized. In this study, we characterize the function of leukocytes including natural killer (NK) cells, macrophages and CAR T cells from transgenic mice expressing a CAR under the control of the pan-hematopoietic promoter, vav, and determine the ability of these mice to respond to ERB expressing tumors. We demonstrate the anti-tumor functions of leukocytes, including antigen specific cytotoxicity and cytokine secretion. The adoptive transfer of CAR T cells provided a greater survival advantage in the E0771ERB tumor model than their wildtype (WT) counterparts. In addition, CAR NK cells and CAR T cells also mediated increased survival in the RMAERB tumor model. When challenged with Her2 expressing tumors, F38 mice were shown to mount an effective immune response, resulting in tumor rejection and long-term survival. This was shown to be predominantly dependent on both CD8(+) T cells and NK cells. However, macrophages and CD4(+) T cells were also shown to contribute to this response. Overall, this study highlights the use of the vav-CAR mouse model as a unique tool to determine the anti-tumor function of various immune subsets, either alone or when acting alongside CAR T cells in adoptive immunotherapy.

Immunotherapy is widely accepted as a powerful new treatment modality for the treatment of cancer. The most successful form of immunotherapy to date has been the blockade of the immune checkpoints PD-1 and CTLA-4. Combining inhibitors of both PD-1 and CTLA-4 increases the proportion of patients who respond to immunotherapy. However, most patients still do not respond to checkpoint inhibitors, and prognostic biomarkers are currently lacking. Therefore, a better understanding of the mechanism by which these checkpoint inhibitors enhance antitumor immune responses is required to more accurately predict which patients are likely to respond and further enhance this treatment modality. Our current study of two mouse tumor models revealed that CD4

Immunotherapy is rapidly emerging as a cancer treatment with high potential. Recent clinical trials with anti-CTLA-4 and anti–PD-1/PD-L1 antibodies (mAbs) suggest that targeting multiple immunosuppressive pathways may significantly improve patient survival. The generation of adenosine by CD73 also suppresses antitumor immune responses through the activation of A2A receptors on T cells and natural killer (NK) cells. We sought to determine whether blockade of A2A receptors could enhance the efficacy of anti–PD-1 mAb. The expression of CD73 by tumor cells limited the efficacy of anti–PD-1 mAb in two tumor models, and this was alleviated with concomitant treatment with an A2A adenosine receptor antagonist. The blockade of PD-1 enhanced A2A receptor expression on tumor-infiltrating CD8+ T cells, making them more susceptible to A2A-mediated suppression. Thus, dual blockade of PD-1 and A2A significantly enhanced the expression of IFNγ and Granzyme B by tumor-infiltrating CD8+ T cells and, accordingly, increased growth inhibition of CD73+ tumors and survival of mice. The results of our study indicate that CD73 expression may constitute a potential biomarker for the efficacy of anti–PD-1 mAb in patients with cancer and that the efficacy of anti–PD-1 mAb can be significantly enhanced by A2A antagonists. We have therefore revealed a potentially novel biomarker for the efficacy of anti–PD-1 that warrants further investigation in patients. Because our studies used SYN-115, a drug that has already undergone phase IIb testing in Parkinson disease, our findings have immediate translational relevance for patients with cancer. Cancer Immunol Res; 3(5); 506–17. ©2015 AACR.

Chimeric antigen receptor (CAR) T cells have been highly successful in treating hematological malignancies, including acute and chronic lymphoblastic leukemia. However, treatment of solid tumors using CAR T cells has been largely unsuccessful to date, partly because of tumor-induced immunosuppressive mechanisms, including adenosine production. Previous studies have shown that adenosine generated by tumor cells potently inhibits endogenous antitumor T cell responses through activation of adenosine 2A receptors (A2ARs). Herein, we have observed that CAR activation resulted in increased A2AR expression and suppression of both murine and human CAR T cells. This was reversible using either A2AR antagonists or genetic targeting of A2AR using shRNA. In 2 syngeneic HER2+ self-antigen tumor models, we found that either genetic or pharmacological targeting of the A2AR profoundly increased CAR T cell efficacy, particularly when combined with PD-1 blockade. Mechanistically, this was associated with increased cytokine production of CD8+ CAR T cells and increased activation of both CD8+ and CD4+ CAR T cells. Given the known clinical relevance of the CD73/adenosine pathway in several solid tumor types, and the initiation of phase I trials for A2AR antagonists in oncology, this approach has high translational potential to enhance CAR T cell efficacy in several cancer types.

Adoptive immunotherapy utilising chimeric antigen receptor (CAR) T cells has demonstrated high success rates in hematological cancers, with up to 90% complete remission being reported in patients with acute lymphoblastic leukemia (ALL). However, this success is not currently transferrable to solid malignancies due in part to the immunosuppressive tumor microenvironment limiting CAR T cell responses. Given that activation of the 4-1BB (CD137) pathway using an agonistic α-4-1BB antibody is known to provide strong co-stimulatory signals for augmenting and diversifying T cell responses, we hypothesized that the combination of α-4-1BB and CAR T cell therapy would result in improved anti-tumor responses. Using a human-Her2 self-antigen mouse model, we found that α-4-1BB significantly enhanced CAR T cell efficacy directed against the Her2 antigen in two different established solid tumor settings. Investigation into the mechanism revealed increased expression of IFNγ and the proliferation marker Ki67 by tumor-infiltrating CAR T cells when combined with α-4-1BB. The importance of IFNγ was demonstrated in vivo where synergistic effects of the combination therapy were abrogated when IFNγ was depleted. Strikingly, we also found that α-4-1BB significantly reduced host immunosuppressive cells at the tumor site including regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs), correlating with an increased therapeutic response. Investigation in immunocompromised RAG -/- mice showed reduced synergistic effects following the combination therapy, suggesting some contribution by host T cells. Furthermore, we found increased frequency of endogenous T cells and dendritic cells in the tumor-draining lymph nodes, supporting potential engagement of host immune cells. We therefore conclude α-4-1BB has a multifunctional role for enhancing CAR T cell responses including engagement of endogenous immune cells, and this combination approach has high translational potential given that CAR T cell therapy has recently been FDA approved for pediatric ALL, and that α-4-1BB is currently being tested in clinical trials against various types of cancer. Citation Format: Sherly Mardiana, Liza B. John, Michael H. Kershaw, Paul A. Beavis, Phillip K. Darcy. A multifunctional role for adjuvant anti-4-1BB therapy in augmenting antitumor responses by CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1530.

Tumors are associated with a microenvironment that can influence responses to immunotherapy. Advanced stages of cancer often involve multiple tumors in different locations in the body. We have demonstrated that the tumor microenvironment can vary with anatomical site, and that normal tissue surrounding the site of tumor implantation can contribute to sculpting the subsequent tumor microenvironment. Furthermore, we demonstrate that tumor responses to immunotherapy, consisting of three agonist antibodies, vary with anatomical site. We also investigated whether tumors and their disparate microenvironment can interact with each other at a distance, in a multiple tumor setting, through a form of cross-talk, to affect their responses to immunotherapy. Our study investigated the cross-talk between two tumors with disparate microenvironments in a mouse model. We demonstrated that immunosuppressive visceral tumors could influence distant subcutaneous (SC) tumors, normally responsive to immunotherapy, to render them resistant. We observed distinct modifications in the SC tumor microenvironment following cross-talk with kidney tumors, which exhibit a type-2 macrophage-related immunosuppressive microenvironment. Indeed, when a concomitant kidney tumor was present in the mouse, the SC tumors were highly infiltrated with M2 macrophages and had a reduced T cell and NK cell effector immune profile. Finally, blocking the M2-associated chemokine CCL2 or depleting macrophages, significantly improved the effect of immunotherapy on SC tumors in the presence of concomitant kidney tumors. This work presents a novel finding describing the potential negative influence that a tumor, with a strong immunosuppressive microenvironment, can exert on distant tumors that would normally be responsive to treatment. This report may lead to a new outlook in the selection and prioritization of treatment of multiple tumors in advanced metastatic cancer, which will consider the anatomical location and corresponding microenvironment of individual tumors. Citation Format: Christel Devaud, Jennifer A. Westwood, Liza John, Carmen S.M. Yong, Paul A. Beavis, Linda A. Snyder, Reto A. Schwendener, Darcy K. Phillip, Michael H. Kershaw. Cross-talk between tumors can affect responses to therapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A105.

In many cancers, regulatory T cells (Treg) play a crucial role in suppressing the effector immune response thereby permitting tumor development. Indeed, in mouse models, their depletion can promote the regression of tumors of various origins, including renal cell carcinoma when located subcutaneous (SC). In the present study, we aimed to assess the importance of Treg immunosuppression in the physiologic context of metastatic renal carcinoma (Renca) disease. To that purpose we inoculated renal tumors orthotopically, intra-kidney (IK), in mice. Treg depletions were performed using anti-CD4 antibody in wild type mice or diphtheria toxin (DT) in Foxp3DTR transgenic mice. Our main observation was that Treg were not the key immunosuppressive component of the IK tumoral microenvironment, compared to the same tumors located SC. We demonstrated that the CD8+ effector immune response was still suppressed in IK tumors when compared to SC tumors, following Treg depletion. Furthermore, the level of program cell death protein (PD)-1 was increased on the surface of CD4+ T cells infiltrating IK tumors compared to SC tumors. Finally, the Treg-independent immunosuppression, occurring in IK tumors, was potent enough to inhibit regression of concomitant SC tumors, normally responsive to Treg depletion. Our findings provide further insight into the immunosuppressive nature of the immune response generated in the kidney microenvironment, suggesting that it can have additional mechanisms in addition to Treg. These observations might help to identify better targets from the kidney tumor microenvironment for future cancer therapies.